Although previous studies have suggested associations between plasma palmitoleic acid and coronary heart disease (CHD) risk factors, including blood pressure, inflammation, and insulin resistance, little is known about the relation of palmitoleic acid and CHD. This ancillary study of the Physicians' Health Study was designed to examine whether red blood cell (RBC) membrane cis-palmitoleic acid and cis-vaccenic acid—2 fatty acids that can be synthesized endogenously—are associated with CHD risk. We used a risk set sampling method to prospectively select 1,000 incident CHD events and 1,000 matched controls. RBC membrane fatty acids were measured using gas chromatography. The CHD cases were ascertained using an annual follow-up questionnaire and validated by an End Point Committee through a review of the medical records. In a conditional logistic regression analysis adjusting for demographics, anthropometric, lifestyle factors, and co-morbidity, the odds ratios and 95% confidence intervals (CIs) for CHD were 1.0 (referent), 1.29 (95% CI 0.95 to 1.75), 1.08 (95% CI 0.78 to 1.51), 1.25 (95% CI 0.90 to 1.75), and 1.48 (95% CI 1.03 to 2.14) across consecutive quintiles of RBC membrane cis-palmitoleic acid (p for trend = 0.041). The odds ratio associated with each SD higher RBC membrane cis-palmitoleic acid level was 1.19 (95% CI 1.06 to 1.35) in a multivariate-adjusted model. Finally, RBC membrane cis-vaccenic acid was inversely associated with CHD risk (odds ratio 0.79, 95% CI 0.69 to 0.91, per SD increase). In conclusion, our data showed a positive association between RBC membrane cis-palmitoleic acid and CHD risk in male physicians. Furthermore, RBC membrane cis-vaccenic acid was inversely related to CHD.